The sex determination mechanism for the Chinese soft-shelled turtle (Pelodiscus sinensis) is subject to controversy. Some populations have been shown to possess sex chromosomes and thus genotypic sex determination (GSD), while others were reported to exhibit temperature-dependent sex determination (TSD). To test whether TSD and GSD coexist in this species or whether populations differ in their sex-determining system, we conducted egg incubation experiments to investigate how temperature influences hatchling sex in a wide range of populations of this species in China. In parallel, we used comparative genome hybridization (CGH) to study the micro-sex chromosomes of adult P. sinensis in the 2 populations that were previously identified to be TSD. The incubation experiments showed that temperature did not affect hatchling sex in any of the studied populations. CGH indicated that turtles have micro-sex chromosomes of the female heterogametic (ZZ/ZW) system in the 2 disputed populations. These results indicate that P. sinensis is a GSD rather than a TSD species. Thus, the apparent coexistence of TSD and GSD in this species is the result of previous misdiagnosis in purportedly TSD populations.

BackgroundInfection of SARS-CoV-2 may cause acute respiratory syndrome. It has been reported that SARS-CoV-2 nucleocapsid protein (N-protein) presents early in body fluids during infection. The direct involvement of N-protein in lung injury is poorly understood.MethodsRecombinant N-protein was pretreated with polymyxin B, a lipopolysaccharide (LPS)-neutralizing agent. C57BL/6, C3H/HeJ (resistant to LPS), and C3H/HeN (control for C3H/HeJ) mice were exposed to N-protein via intratracheal administration to examine acute lung injury. In vitro, bone marrow–derived macrophages (BMDMs) were cultured with N-protein to study phosphorylation of nuclear factor kappa B (NF-ĸB) p65, macrophage polarization, and expression of proinflammatory cytokines.ResultsN-protein produced acute lung injury in C57BL/6 mice, with elevated protein permeability, total cell count, neutrophil infiltration, and proinflammatory cytokines in the bronchioalveolar lavage. N-protein also induced lung injury in both C3H/HeJ and C3H/HeN mice, indicating that the effect could not be attributed to the LPS contamination. N-protein triggered phosphorylation of NF-ĸB p65 in vitro, which was abolished by both N-protein denaturation and treatment with an antibody for N-protein, demonstrating that the effect is N-protein specific. In addition, N-protein promoted M1 macrophage polarization and the expression of proinflammatory cytokines, which was also blocked by N-protein denaturation and antibody for N-protein. Furthermore, N-protein induced NF-ĸB p65 phosphorylation in the lung, while pyrrolidine dithiocarbamate, an NF-ĸB inhibitor, alleviated the effect of N-protein on acute lung injury. ConclusionsSARS-CoV-2 N-protein itself is toxic and induces acute lung injury in mice. Both N-protein and NF-ĸB pathway may be therapeutic targets for treating multi-organ injuries in Coronavirus disease 2019 (COVID-19).

Despite widespread temperature-dependent sex determination (TSD) in reptiles, it is still unclear how the molecular network responds to temperature variation and drives the sexual fate. Profiling of sex-related genes is the first step in understanding the sex determination system in reptiles. In this study, we cloned the full-length coding sequences of Cyp19a1, Foxl2, Rspo1, Sf1, and Sox9 in an Asian freshwater turtle (Mauremys reevesii) with TSD and identified the expression patterns of these genes and Dmrt1 at different incubation temperatures to understand their roles in urogenital development. Our results showed that Cyp19a1, Foxl2, and Rspo1 were expressed in the adrenal-kidney-gonadal complex at a high level in females, while Sf1 and Dmrt1 were highly expressed in males. In addition, Foxl2 and Rspo1 showed sex-dimorphic expression in the presumed early thermosensitive period (TSP), Dmrt1 was upregulated at the beginning of the presumed TSP, and Sox9 did not show sex-dimorphic expression until the end of the presumed TSP. These results suggest that Foxl2 and Rspo1 are probably upstream genes involved in female sex determination and that Dmrt1 may be a key factor in male sex determination. Therefore, our study provides a solid foundation for further investigations on the molecular mechanism underlying sex determination in M. reevesii.